Important biological functions including maintenance of homeostasis in the living body, reproduction, development of individuals, metabolism, growth, control of the nervous, circulatory, immune, digestive or metabolic system, sensory adaptation, etc. are regulated by cells that receive endogenous factors such as various hormones and neurotransmitters or sensory stimulation such as light or odor, via specific receptors present on cell membranes reserved for these factors or stimulation and interact with them. Many of these receptors for hormones or neurotransmitters by such functional regulation are coupled to guanine nucleotide-binding proteins (hereinafter, sometimes merely referred to as G proteins), and are characterized by developing a variety of functions through intracellular signal transduction via activation of the G proteins. In addition, these receptor proteins possess common seven transmembrane domains. For the reasons above, these receptors are therefore collectively referred to as G protein-coupled receptors or seven transmembrane receptors. As stated above, it is known that various hormones or neurotransmitters and their receptor proteins are present and interact with each other to play important roles for regulating the biological functions. However, it is even now poorly understood if any other unknown substances (hormones, neurotransmitters, etc.) and receptors to these substances are present.
In recent years, the human gene has been clarified at an accelerating pace by accumulated sequence information through sequencing of human genomic DNA or various human tissue-derived cDNA at random and rapid progress in gene analysis technology. Based on the foregoing, it is manifested that there are many genes supposed to encode proteins with unknown functions. G protein-coupled receptors not only have seven transmembrane domains but many common sequences are present in their nucleic acids or amino acids, and can thus be clearly identified to be G protein-coupled receptors in such proteins. On the other hand, such G protein-coupled receptor genes are obtained by polymerase chain reaction (hereinafter abbreviated as PCR) utilizing such a similarity in structure (Nature Cell Biology, 2, 703-708 (2000)). In these G protein-coupled receptors thus obtained so far, ligands to some receptors that are subtypes having high homology in structure to known receptors may be readily predictable but in most cases, their endogenous ligands are unpredictable so that no ligands corresponding to these receptors are found. For this reason, these receptors are called orphan receptors. It is likely that unidentified endogenous ligands to such orphan receptors would take part in the biological phenomena poorly analyzed, because the ligands were unknown. And when such ligands are associated with important physiological effects or pathologic conditions, it is expected that development of these receptor agonists or antagonists will result in breakthrough of new drugs (Stadel, J. et al., TiPS, 18, 430-437, 1997; Marchese, A. et al., TiPS, 20, 370-375, 1999; Civelli, O. et al., Brain Res., 848, 63-65, 1999). Until now, however, there are few examples to actually identify ligands to orphan G protein-coupled receptors.
Recently, some groups attempted to investigate ligands to these orphan receptors and reported isolation of ligands, which are novel physiologically active peptides, and determination of their structures. Reinsheid et al. and Meunier et al. independently introduced cDNA encoding orphan G protein-coupled receptor LC132 or ORL1 into animal cells to express a receptor, isolated a novel peptide from porcine brain or rat brain extract using response of the receptor as an indicator, which was named orphanin FQ or nociceptin with reference to its response, and determined its sequence (Reinsheid, R. K. et al., Science, 270, 792-794, 1995; Meunier, J.-C. et al., Nature, 377, 532-535, 1995). This peptide was reported to be associated with a sense of pain. Further investigations on the receptor in knockout mouse revealed that the peptide was involved in memory (Manabe, T. et al., Nature, 394, 577-581, 1998).
Subsequently, novel peptides including PrRP (prolactin releasing peptide), orexin, apelin, ghrelin and GALP (galanin-like peptide), etc. were isolated as ligands to orphan G protein-coupled receptors (Hinuma, S. et al., Nature, 393, 272-276, 1998; Sakurai, T. et al., Cell, 92, 573-585, 1998; Tatemoto, K. et al., Bichem. Biophys. Res. Commun., 251, 471-476, 1998; Kojima, M. et al., Nature, 402, 656-660, 1999; Ohtaki, T. et al., J. Biol. Chem., 274, 37041-37045, 1999).
On the other hand, some receptors to physiologically active peptides hitherto unknown are unraveled by similar methods. It was unraveled that a receptor to motilin associated with contraction of intestinal tracts was GPR38 (Feighner, S. D. et al., Science, 284, 2184-2188, 1999). In addition, SLC-1 was identified to be a receptor to melanin-concentrating hormone (MCH) (Chambers, J. et al., Nature, 400, 261-265, 1999; Saito, Y. et al., Nature, 400, 265-269, 1999; Shimomura, Y. et al., Biochem. Biophys. Res. Commun., 261, 622-626, 1999; Lembo, P. M. C. et al., Nature Cell Biol., 1, 267-271, 1999; Bachner, D. et al., FEBS Lett., 457, 522-524, 1999). Also, GPR14 (SENR) was reported to be a receptor to urotensin II (Ames, R. S. et al., Nature, 401, 282-286, 1999; Mori, M. et al., Biochem. Biophys. Res. Commun., 265, 123-129, 1999; Nothacker, H.-P. et al., Nature Cell Biol., 1, 383-385, 1999, Liu, Q. et al., Biochem. Biophys. Res. Commun., 266, 174-178, 1999). It was shown that MCH took part in obesity since its knockout mice showed hypophagic and lean phenotype (Shimada, M. et al., Nature, 396, 670-674, 1998), and because its receptor was unraveled, it became possible to explore a receptor antagonist likely to be useful as an anti-obesity agent. It is further reported that urotensin II shows a potent action on the cardiocirculatory system, since it induces heart ischemia by intravenous injection to monkey (Ames, R. S. et al., Nature, 401, 282-286, 1999).
As described above, orphan receptors and ligands thereto are often engaged in a new physiological activity, elucidation of which will lead to development of novel drugs. However, investigations of ligands to orphan receptors are accompanied by many difficulties. While the presence of many orphan receptors was unraveled to date, only a very few ligands were discovered for these receptors.
The present inventors found a novel receptor ZAQ, which is an orphan G protein-coupled receptor (a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 of the specification: hereinafter sometimes simply referred to as ZAQ in the specification). However, it was heretofore unknown what the ligand was.
It has been the problem to find a ligand to the orphan receptor protein ZAQ and establish a method of screening a compound characterized by using ZAQ and a ligand thereto.